The present invention relates generally to recombinant DNA technology and, particularly, to methods for generating combinatorial macromolecule libraries and screening these libraries for peptides of interest via genetic selection.
The identification and isolation of proteins involved in cellular regulatory control is fundamental to the understanding of a wide variety of cellular mechanisms. As many pathologies, such as oncogenesis, involve a breakdown of these regulatory mechanisms, the isolation of these proteins is a significant step in the process of discovering new therapeutics. One means of identifying these proteins involves the generation of peptides which inhibit regulatory mechanisms by acting as competitors in the normal protein-protein interactions, the interactions between proteins and nucleic acids, the interactions between proteins and small molecules as well as other intracellular molecular interactions. Peptides having a large number of applications of use, such as therapeutic or diagnostic reagents, may thus be identified without any prior information on the structure of the expected ligand or receptor.
Advances in molecular biology make possible the construction of extremely large collections of peptide sequences as potential inhibitors of the cell cycle and other biological pathways. The generation of large numbers of peptide sequences by the cloning and expression of randomly-generated mixtures is possible in the appropriate recombinant vectors (Oliphant et al. (1986), Gene 44:177-183). Such a large number of peptides can be produced, however, that methods for efficient physical and genetic selection are required. Without flexible methods of analysis, the usefulness of these large peptide libraries in identifying molecules of interest may be lost.
A number of systems for screening proteins and polypeptides have been described. The fusion phage approach can be used to screen proteins (Parmley and Smith, (1988), Gene 73:305-318). Others have described phage-based systems in which the peptide is fused to the pIII coat protein of filamentous phage (Scott and Smith, (1990), Science 249:386-390; Devlin et al., (1990), Science 249:404-406; and Cwirla et al., (1990), Proc. Natl. Acad. Sci. USA 87:6378-6382). Others have also devised combinatorial libraries in other systems. Colas et al. describes the expression of a combinatorial library of peptides displayed in the active loop of E. Coli thioredoxin and the use of a two-hybrid system to select those that bind human Cdk2 (Colas et al., (1996), Nature 380:548-560).
While this art is well developed, a need remains for methods of constructing and screening macromolecule libraries, in addition to those described in the art. For example, the screening methods of this art are limited to and involve the use of assays such as peptides fused to biologically active carrier proteins or fusion proteins compatible with the specific biological activities. In addition, the above methods are limited in that they do not provide flexibility in using macromolecules that can be expressed and genetically selected intracellularly; yet such peptides would add great diversity to the proteins and processes that may be targeted by such libraries.
In contrast to the prior art, novel macromolecule libraries and strategies for identifying peptide inhibitors of cell cycle control and other biological pathways and the targets they inhibit are described herein. The power of this technology is fourfold: it requires no prior assumptions about the biochemical nature of the target pathway; it identifies those members of a pathway that are targets for inhibition by small molecules; weak initial inhibitors can be evolved by mutation and selection into potent inhibitors; and information from this process provides valuable structure-function information to assist in peptidomimetic chemistry to produce drugs directed against cancer and other diseases.
Cancer therapy attempts to kill tumor cells while sparing normal cells. Current therapies use agents that are broadly toxic to dividing cells, make patients profoundly ill, and are only effective against a minority of cancers. Over the last 10 years, three important findings have dramatically improved prospects for more effective chemotherapy. First, cancer is now understood to be a very diverse disease, with different patterns of genetic alterations in different tumors. Second, all cancers show enormous genetic instability due to lesions in the fundamental processes of DNA replication, DNA repair and chromosome segregation; or inactivation of the cell cycle checkpoints that coordinate the events of the cell cycle with each other. Third, the DNA repair, DNA replication, chromosome segregation, and checkpoint pathways have been conserved during evolution, meaning that conclusions from work on simple eukaryotes are directly applicable to humans. It is now possible to exploit these advances by using cells such as budding yeast to launch novel programs of drug discovery that find agents which selectively kill tumors with specific molecular lesions, and have low toxicity towards normal cells.
The present invention provides methods for generating and screening via genetic selection random libraries of macromolecules to identify sequences that interact with target molecules of interest. The libraries described herein include peptide chimeras (also referred to herein as a peptide library), wherein an amino acid sequence is inserted into a carrier molecule, e.g., a protein backbone. The macromolecules so identified (chimeras or the peptide portion of the chimeras) can be used for therapeutic, diagnostic and related purposes by interacting with a target protein of interest to inhibit or promote the biological activity of that protein. The chimeras can also be used to identify the targets to which they bind, thus providing important information for the development of small molecule inhibitors of the same targets.
The invention further provides a screening method which comprises the steps of (a) growing the host cells under conditions which genetically select for clones that contain peptides that have the ability to disrupt the interactions between molecules that affects a biological process and (b) isolating the vectors that encode the genetically selected peptides. By repeating the affinity selection process one or more times, the plasmids encoding the peptides of interest can be enriched. By increasing the stringency of the selection, e.g., by decreasing the expression of the chimeras, increasing the temperature or varying other medium conditions, peptides of increasingly higher affinity can be identified.
The invention also concerns methods wherein peptides of interest can be mutagenized to create inhibitors of varying affinities. The invention further concerns methods wherein a biological target of the peptide inhibitor can be identified. For inhibitors specific to well characterized pathways, the overexpression of specific members of the pathways can be used to overcome the inhibition and thereby identify the peptide target.